Scale bar, 5 m. endosomes CY3 where the PS-binding protein EHD1 is recruited to facilitate endosome fission. Our study highlights a novel lipid exchange at ERCendosome MCSs as a nonenzymatic PI4P-to-PS conversion mechanism that organizes membrane remodeling during retrograde membrane trafficking. Introduction Membrane contact sites (MCSs) are sites of direct communication for organelles and the plasma membrane (PM), where two cellular membranes are closely apposed (Scorrano et al., 2019). Accumulating evidence demonstrates that the ER forms MCSs with most of the organelles or the PM in eukaryotes (Bohnert, 2020; Wu et al., 2018). MCSs have been shown to be involved in several functions, including but not limited to Ca2+ regulation, lipid metabolism, and signaling (Prinz et al., 2020). In particular, MCSs serve as a zone for nonvesicular lipid transport mediated by so-called lipid transfer proteins (LTPs; Wong et al., 2019). LTPs generally possess a hydrophobic pocket that harbors lipid molecules, and they act as regulators for intracellular lipid distribution by transferring lipids between cellular membranes (Holthuis and Menon, 2014). Oxysterol-binding protein (OSBP)Crelated proteins (ORPs) are a class of LTPs that consist of 12 different members in mammals (Raychaudhuri and Prinz, 2010). ORPs have the well-conserved OSBP-related domain (ORD), a ligand-binding pocket that accommodates a lipid ligand (Tong et al., 2016). Studies now show that the repertoire of their ligands has expanded to include not only oxysterol but also other lipids such as cholesterol, phosphoinositides, phosphatidylserine (PS), or phosphatidylcholine (PC; Balla et al., 2019). Additionally, most ORPs contain a PH domain and an ER localization domain/motif such as a transmembrane domain or the two phenylalanines in acidic tract (FFAT) motif that is recognized by the ER protein vesicle-associated membrane protein (VAMP)Cassociated protein A (VAPA)/VAPB (Alli-Balogun and Levine, 2019). Via those dual membrane association determinants, ORPs localize at MCSs by simultaneously interacting with the ER and other cellular membranes, and they act as a sensor or transporter of their lipid CY3 ligands (Kentala et al., 2016; Pietrangelo and Ridgway, 2018). A characteristic functional property CY3 of ORPs is their lipid countertransport activity. Recent studies suggest that several ORPs harbor two different lipids as their ligands, phosphatidylinositol 4-phosphate (PI4P) as a common ligand or other lipids as another ligand, VHL and mediate counterdirectional transport of those two different lipids (Chung et al., 2015; Mesmin et al., 2013; Sohn et al., 2018; DSouza et al., 2020). We have shown that ORP5 and ORP8 mediate countertransport of PI4P and PS at the ERCPM MCSs (Chung et al., 2015). This lipid countertransport was driven by a PI4P concentration gradient between the ER and the PM. PI4P is continuously synthesized at the PM, while it is metabolically degraded at the ER. The PI4P concentration gradient determines the direction of its transport to the ER, which in turn facilitates the counterdirectional transport of PS from the ER. Therefore, this mechanismPI4P-driven lipid countertransporthelps deliver lipids from the ER to other membranes in exchange for PI4P via MCSs (Antonny et al., 2018; Balla et al., 2019; Mesmin and CY3 Antonny, 2016). Despite the fact that the general mechanism of PI4P-driven lipid countertransport has been characterized, cellular function regulated by this mechanism is still largely unknown. In PI4P-driven lipid countertransport, PI4P is passively replaced by ORPs with other lipids that are transported from the ER. This would be a lipid conversion that changes a property of a membrane and thus potentially impacts numerous cellular functions. However, which cellular processes directly couple to this lipid conversion event and how cellular processes harness such lipid conversion remain elusive. More specifically, how such CY3 exchanged lipid exerts its specific biological function is poorly understood. Answering those questions will reveal the biological significance of PI4P-driven lipid countertransport at MCSs. In this study, we identified ORP10 as a bona fide lipid exchanger at ERCendosome MCSs. ORP10 localized at the MCSs between the ER and the PI4KII-positive endosomes that are tethered by ORP9 and VAP. Both in vitro and in situ, ORP10 mediates exchange of two different lipids, PS and PI4P, the latter of which was identified in this study as a ligand of ORP10. In the cellular context, ORP10 supplied PS in exchange for PI4P to endosomes where the PS-binding protein EHD1 is recruited, thereby promoting the fission process of tubulovesicular carrier from endosomes in the retrograde trafficking pathway. Thus, ORP10 is a new lipid exchanger at ERCendosome MCSs,.